The present invention relates to the use of restriction endonucleases as therapeutic and disinfectant anti-viral agents.
There are currently very few effective anti-viral agents, although the many virally transmitted diseases account for much human suffering and mortality. Thus, there is a great need for safe and effective anti-viral agents that can serve in therapies for these diseases, including life-threatening and fatal diseases such as hepatitis and AIDS.
Bacterial cells long have been known to contain restriction-modification systems that protect them from viral infection. See, for instance, Gingeras, T., "Restriction-Modification Systems" in MODERN MICROBIAL GENETICS 301-321 (Wiley-Liss, Inc. 1991). A restriction-modification system generally operates through two complementing enzymatic activities, an endonucleolytic activity and a modification activity. The endonucleolytic activity involves recognition of a specific sequence in viral DNA and subsequent endonucleolytic cleavage across both strands of the DNA. The modification activity involves the same sequence recognition step followed by modification of a base in the sequence, which interferes with the cleavage activity of the endonuclease. Thus, host cell DNA modified by the endogenous modification enzyme, is protected from degradation by the restriction enzyme which destroys the unprotected DNA of infecting virus.
Restriction-modification systems like those of prokaryotes have not been found as pervasively in eukaryotic cells, although restriction endonucleases have been isolated from several eukaryotic sources. See Sklar et al. (1986) J. Biol. Chem. 261: 6806-6810 (Chlamydomonas reinhardtii) and Lao et al. (1986) Sci. Sin. (Series B) 29: 947-953 (human). The genomic DNA of eukaryotic cells contains modified bases, however these modifications differ in many ways from those of prokaryotes and perform functions unrelated to viral infection. Viruses that infect eukaryotic cells do not encounter the same endonucleolytic defenses that beset viruses invading prokaryotic cells.
Nonetheless, many viruses which infect eukaryotic cells possess, during at least one part of their life cycle, genomes that consist of double stranded DNA which can be cleaved readily by restriction endonucleases derived from a prokaryotic source. Furthermore, it is well-known that restriction endonuclease cleavage can prevent viral DNA, such as bacteriophage lambda DNA, from packaging to form infectious virus particles, and can also destroy the ability of DNA to transform cells in vitro using, for instance, calcium phosphate precipitation. Accordingly, there has been some speculation that providing a restriction-modification system to eukaryotic cells might provide a means to prevent viral infection.
Strategies for achieving this end heretofore have focused on introducing into eukaryotic cells genes that provide the endonuclease and the modification activities. Providing the modification activity was thought necessary to protect cellular DNA from being degraded by the restriction enzyme, i.e., to distinguish cellular and viral DNA so that the endonucleolytic activity would not degrade the cellular DNA. Progress along this avenue has not been promising, however.
For instance, Kwoh expressed the bacterial PaeR7 methylase gene in mouse Ltk cells to study the effect of PaeR7 methylation on replication and transcription of eukaryotic DNA. Kwoh et al., Proc. Natl. Acad. Sci. USA 83: 7713 (1986). Kwoh noted that cell lines expressing the PaeR7 methylase would be suitable to the introduction of the PaeR7 endonuclease and that cells with both enzymes would contain a restriction-modification system that might render them resistant to viral infection.
Subsequently, Kwoh studied the expression of both methylase and endonuclease in eukaryotic cells and assessed the ability of the exogenously derived restriction-modification system to retard viral infection. Kwoh et al., Nuc. Acids Res. 16: 11489 (1988). Kwoh did not observe increased resistance to infection by murine adenovirus, herpes simplex virus, or vaccinia virus in any of the cell lines that expressed the bacterial endonuclease, even though the enzyme was expressed. Thus, this particular approach to using restriction enzymes as antiviral agents has not been successful.
In attempting to explain the negative results, Kwoh speculated that the methylase in these experiments might have modified not only the genomic but also the viral DNA, thus preventing the endonuclease from affecting the viral DNA. Alternatively, eukaryotic glycosylation of a bacterial restriction enzyme might have interfered with its ability to cleave viral DNA. In this regard, glycosylation could affect the endonucleolytic activity directly, the ability of the enzyme to bind the DNA substrate, or the ability of the enzyme to penetrate the viral nucleocapsid to cleave viral DNA in situ.
In any event, the failure of restriction endonuclease expressed by eukaryotic cells to inhibit viral infection has militated against the possibility that restriction enzymes might be employed somehow to prevent or ameliorate viral infection of eukaryotic cells.